Method of determining energy transfer efficiency of a donor-acceptor pair of fluorescent dyes

Abstract

The energy transfer efficiency of a donor-acceptor pair of fluorescent dyes can be determined by first measuring the fluorescence and absorption spectra of donor-protein, acceptor-protein, mixture of donor-protein and acceptor-protein, and donor-acceptor-protein conjugates, then separating the respective spectra into their respective donor-protein complex and acceptor-protein complex components using multiple linear regression, and then determining the transfer efficiency on the basis of the quenching of the donor fluorescence based on the spectral data thus obtained.

We claim: 
     
       1. A method of determining the energy transfer efficiency of a donor-acceptor pair of fluorescent dyes useful in fluorescent energy transfer immunoassay which comprises: (a) measuring the fluorescence and absorption spectra of donor-protein, acceptor-protein, mixture of donor-protein and acceptor-protein and donor-acceptor-protein conjugates;   (b) separating the respective spectra measured in (a) into their respective donor-protein complex and acceptor-protein complex components using multiple linear regression and employing models   F.sub.a-d =α·F.sub.d +β·F.sub.a +ε     and     A.sub.a-d =α'·A.sub.d +β'·A.sub.a +ε'        where F d , F a , and F a-d  are the fluorescence spectra of the donor-protein conjugate, the acceptor-protein conjugate, and the donor-acceptor-protein conjugate, respectively, α and β are the linear coefficients to be determined and ε is the error term and A d , A a , and A a-d  are the absorption spectra of the donor-protein conjugate, the acceptor-protein conjugate, and the donor-acceptor-protein conjugate, respectively, α' and β' are the linear coefficients to be determined and ε' is the error term; and   (c) determining the energy transfer efficiency on the basis of quenching of donor fluorescence using the equation ##EQU2##  where E is energy transfer efficiency, F d  is the fluorescence intensity of donor in the absence of acceptor, A d  is the absorbance of donor in the absence of acceptor, F a   d  is the fluorescence intensity of the donor in the presence of acceptor, and A a   d  is the absorbance of donor in the presence of acceptor.   
     
     
       2. A method as claimed in Claim 1 wherein the interaction components between donor and acceptor are also determined by multivariate factor analysis and these interaction components are incorporated into the step (b) multiple linear regression. 
     
     
       3. A method as claimed in claim 2 wherein the interaction components between donor and acceptor was determined by multivariate factor analysis using a data matrix consisting of fluorescence spectra of the donor-protein, acceptor-protein, mixture of donor-protein and acceptor-protein, and donor-acceptor-protein conjugates.